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The Preparation of αvβ3 Integrin Targeted Super Paramagnetic Liposome and Tumor Vasculature Targeted MR Imaging

Author: QuHaiYuan
Tutor: XuKe
School: China Medical University
Course: Medical Imaging and Nuclear Medicine
Keywords: super-paramagnetic liposome RGD peptide αvβ3-integrin angiogenesis SPIO Vx2
CLC: R730.4
Type: PhD thesis
Year: 2008
Downloads: 391
Quote: 1
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Abstract


Purposeαvβ3-integrin is strongly over expressed on angiogenic endothelial cells within tumors,and plays key roles in tumor angiogenesis.It can be looked as a tumor specific marker.Thus,this localized tissue distribution ofαvβ3 integrins can potentially enable selective contrast agent delivery into neo-vasculature by usingαvβ3 targeted drug delivery systems.As a result,we can detect and diagnosis tumor in a more early and precise way.In this study,an angiogenic endothelial cell-targeted contrast agent delivery system was developed by attaching RGD peptide on the surface of liposome and enveloping SPIO into the inner aqueous phase.Its ability to detect tumor angiogenesis was evaluated by in vitro fluorescence microscopy and in vivo MR molecular imaging.Materials and methodsTwo different liposome preparations,targeted RGD-super paramagnetic liposome and non-targeted super paramagnetic liposome,were prepared.The targeted liposome was prepared by conjugating RGD to the distal ends of lipid-anchored PEG-chains present in the liposome membrane.Liposome characterization,including particle size and size distribution,envelop ratio of SPIO and relaxation ratio were measured.Human umbilical vein endothelial cells(HUVEC) about 106 were transferred to FACS tubes and incubated with the two liposome preparations for 1 hour at 37℃; another group HUVEC was added only with PBS.In the competitive experiment, HUVEC were incubated with tenf61d concentration free RGD,subsequently,RGD fluorescent liposome were added and incubated with HUVEC.Cells were analyzed by flow cytometry.For Confocal laser scanning microscopy(CLSM) analysis,HUVEC were grown on cover slips to 70%confluence and incubated with calcein labeled RGD-liposome and non-targeted liposome diluted in culture medium for 1 hour at 37℃or 4℃. Fluorescent images of cells were analyzed using a TCS SP2 confocal microscope.In vivo MRI molecular imaging study was carried out on Vx2 tumor bearing rabbit.Tumor-bearing rabbit were prepared by inoculating Vx2 subcutaneously on the both flank.Tumor bearing rabbits were injected I.V with the two liposome preparations on concentration of 3mg Fe/kg body weight.Before and after injection,MRI scanning was performed several time,MRI scanning was finished 24 hour later.The signal intensities of the tumor pre-and post-contrast agent injection were compared,the signal intensities difference between tumor and muscle were also compared.The Contrast-to-noise Ratios(CNR) of tumor and muscle were compared.The rabbits were then sacrifice and the tumor were excised and used for histological analysis.The slice were stained with hematoxylin-eosin and Perls Prussian and examined with bright light microscopy.ResultsThe mean size of targeted RGD-super paramagnetic liposome and non-targeted super paramagnetic liposome are 144/147nm separately.The Packaging Ratio of targeted RGD-super paramagnetic liposome and non-targeted super paramagnetic liposome are 0.27mg/ml and 0.28mg/ml.The conjugation ratio for RGD to liposome is 74.97 percent.The r2 relaxivities of targeted liposome is 1769 mM-1s-1,1356 mM-1s-1 for non-targeted liposome.The r1 relaxivities is 108 mM-1 s-1,76 mM-1 s-1 for targeted and non-targeted liposome.Transmission electron microscope(TEM) study revealed SPIO in the inner aqueous phase. The FACScan flow cytometry revealed the ratio of positive stained cell are 76.15%, 10.81%,18.96%and 0.37%separately for targeted,non-targeted,competitive group and blank control group.The targeted group was significantly higher than the others (P<0.002).The geometric mean fluorescence intensity of targeted group(128.71) was significantly higher than the non-targeted group(36.71) and competitive group(39.78)(P<0.001).For Confocal laser scanning microscopy analysis,the fluorescence localized on the surface of liposome at 4℃,and in the cytoplasm at 37℃for the targeted group.There was no fluorescence on the non-targeted group.The in vivo magnetic resonance imaging revealed the signal intensity and CNR of tumor drop after the contrast agent injection.At the time point of 15 hour,the CNR drop to the bottom for the targeted group.And for the non-targeted group,the CNR bottom out at the time point of 2 hour after injection.ConclusionThe RGD targeted super-paramagnetic liposome we prepared got high relaxivities. It can bind withαvβ3 integrins selectively and can be detected by in vivo MR imaging.

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CLC: > Medicine, health > Oncology > General issues > Tumor diagnostics
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